Versatile transformation system that is applicable to both multiple transgene expression and gene targeting for thraustochytrids

Keishi Sakaguchi, Takanori Matsuda, Takumi Kobayashi, Jun Ichiro Ohara, Rie Hamaguchi, Eriko Abe, Naoki Nagano, Masahiro Hayashi, Mayumi Ueda, Daiske Honda, Yuji Okita, Yousuke Taoka, Shinichi Sugimoto, Nozomu Okino, Makoto Ito

Research output: Contribution to journalArticlepeer-review

42 Citations (Scopus)

Abstract

A versatile transformation system for thraustochytrids, a promising producer for polyunsaturated fatty acids and fatty acid-derived fuels, was established. G418, hygromycin B, blasticidin, and zeocin inhibited the growth of thraustochytrids, indicating that multiple selectable marker genes could be used in the transformation system. A neomycin resistance gene (neor), driven with an ubiquitin or an EF-1α promoter-terminator from Thraustochytrium aureum ATCC 34304, was introduced into representatives of two thraustochytrid genera, Aurantiochytrium and Thraustochytrium. The neor marker was integrated into the chromosomal DNA by random recombination and then functionally translated into neor mRNA. Additionally, we confirmed that another two genera, Parietichytrium and Schizochytrium, could be transformed by the same method. By this method, the enhanced green fluorescent protein was functionally expressed in thraustochytrids. Meanwhile, T. aureum ATCC 34304 could be transformed by two 18S ribosomal DNA-targeting vectors, designed to cause single- or double-crossover homologous recombination. Finally, the fatty acid Δ5 desaturase gene was disrupted by double-crossover homologous recombination in T. aureum ATCC 34304, resulting in an increase of dihomo-γ-linolenic acid (C20:3n-6) and eicosatetraenoic acid (C20:4n-3), substrates for Δ5 desaturase, and a decrease of arachidonic acid (C20:4n-6) and eicosapentaenoic acid (C20:5n-3), products for the enzyme. These results clearly indicate that a versatile transformation system which could be applicable to both multiple transgene expression and gene targeting was established for thraustochytrids.

Original languageEnglish
Pages (from-to)3193-3202
Number of pages10
JournalApplied and environmental microbiology
Volume78
Issue number9
DOIs
Publication statusPublished - May 2012

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

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